Inhibition of biofilm formation

ABSTRACT

The present invention relates to compositions and methods for reducing or inhibiting biofilm comprising modulating expression of a cysB gene in a cell. The invention also provides methods for modulating the expression of a cysB, cysD, cysI, cysJ, cysK, and ybiK. The invention further provides methods for identifying gene(s) involved in biofilm formation and for identifying biofilm inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication Ser. No. 60/587,680, filed on Jul. 14, 2004, and U.S.provisional patent application Ser. No. 60/609,763, filed on Sep. 14,2004.

FIELD OF THE INVENTION

The invention relates to methods for reducing or inhibiting biofilmformation. The invention also relates to methods for modulating theexpression of a cysB gene. Further, the present invention relates tomethods for identifying genes involved in biofilm formation and foridentifying biofilm inhibitors.

BACKGROUND OF THE INVENTION

Bacterial biofilms exist in natural, medical, and engineeringenvironments. The biofilm may offer a selective advantage to amicroorganism to ensure its survival, or allow it a certain amount oftime to exist in a dormant state until suitable growth conditions arise.This selective advantage could pose serious threats to human health. Forexample, biofilms are involved in 65% of human bacterial infections.Biofilms are also involved in prostatitis, biliary tract infections,urinary tract infections, cystitis, pyelonephritis, lung infections,sinus infections, ear infections, acne, and chronic wounds.

Biofilms contribute to a variety of medical conditions. Each year in theUnited States alone, over 7 million patients receive medical deviceimplants, including central venous catheters, endotracheal tubes,mechanical heart valves, pacemakers, and prosthetic joints.Approximately one-half of these patients develop nosocomial infections,and approximately 80,000 deaths per year are attributed to nosocomialinfections. Biofilms provide a structural matrix that facilitatesbacterial adhesion to the inert surfaces of medical device implants andvenous catheters. Microscopic studies confirm that central venouscatheters are coated by bacteria embedded in biofilms. Unfortunately,more than 1 million patients develop urinary tract infections from suchcatheters.

Some diseased tissues, such as tumors, are susceptible to bacterialcolonization. Bacterial colonization has been identified in calcifiedhuman aneurysms, carotid plaques, femoral arterial plaques, and cardiacvalves. Arterial calcification resembles infectious lesion formation inanimal models of atherosclerosis. A toxin produced by Cag-A positiveHelicobacter pylori colonization of the stomach could lead to tissueinflammation and lesions in the arterial walls resulting inatherosclerosis. Bacterial colonization could also lead to the formationof kidney stones. Eradication of bacteria, and the biofilms that protectthem, from the diseased tissue enables the host's immune system and/or apharmaceutical agent to reach the diseased tissue. For example,clostridia spores and attenuated Salmonella typhimurium, used to delivertherapeutic proteins to tumors, may be more effective if the biofilm didnot exist or is removed.

Biofilms may also cause diseases, such as cystic fibrosis, or contributeto chronic symptoms. Chronic bacterial infections represent a seriousmedical problem in the United States. Antibiotics are typically used totreat both acute and chronic infections. In chronic bacterialinfections, biofilms protect the bacteria from the antibiotics and thehost's immune system, thus increasing the rates of recurring symptomsand resistance to the antibiotics. Researchers theorized that a biofilmgives bacteria a selective advantage by reducing the penetration of anantibiotic to the extent necessary to eradicate the bacteria. Throughbiofilms, the microbes can resist antibiotics at high concentrations,about 1 to 1.5 thousand times higher than necessary in the absence ofbiofilms. Not surprisingly, during an infection, bacteria surrounded bybiofilms are rarely resolved by the host's immune defense mechanisms.

As discussed above, biofilms provide a protective barrier for bacteria,thus, allowing the bacteria to resist antibiotic treatments. Developersof antibiotics must face the continuous challenge of antibioticresistance. Antibiotic resistance significantly hinders treatment of themedical condition. For example, microbial resistance to minocycline andrifampin, which are widely used to treat infections, is emerging. A 1998study of an intensive care unit revealed that 6 out of 7vancomycin-resistant enterococci were resistant to rifampin.

Biofilm inhibition offers numerous advantages. Bacteria have no knownresistance to biofilm inhibitors. Thus, unlike antibiotics, biofilminhibitors can be used repeatedly and effectively in the same patientand for the same medical condition. For example, biofilm inhibitors maybe employed to treat, cure, or prevent acute or chronic infections. Theymay be used to control microorganisms residing on living tissues. Theymay also be used to cure, treat, or prevent arterial damage, gastritis,urinary tract infections, cystitis, otitis media, leprosy, tuberculosis,benign prostatic hyperplasia, chronic prostatitis, chronic infections ofhumans with cystic fibrosis, osteomyelitis, bloodstream infections, skininfections, open wound infections, and any acute or chronic infectionthat involves or possesses a biofilm.

Biofilm inhibitors can act specifically on the biological mechanismsthat provide bacteria protection from antibiotics and from a host'simmune system. In one study of urinary catheters, rifampin was able toclear planktonic or suspended methicillin-resistant Staphylococcusaureus, but was unable to eradicate the bacteria in a biofilm. Currenttreatment of infections, e.g. nosocomial infections, often requiressequential or simultaneous administration of a combination of products,such as amoxicillin/clavulanate and quinupristin/dalfopristin. A directinhibition of the bacterial mechanisms used to form biofilms may helpreduce blood stream infections (BSI).

In addition, a direct inhibition of the bacterial mechanisms used toform biofilms delays the onset of microbial resistance to antibiotics,and possibly, reduces the emergence of multi-resistant bacteria. Anotherapproach to reducing or inhibiting biofilm formation is to applyevolutionary pressure to the bacterial growth mechanisms. Accordingly,extensive research are devoted to elucidating the genes, especially thecritical players, that are involved in controlling biofilm formation.

Accordingly, for the reasons discussed above and others, there continuesto be a need for a means to control biofilm and its formation.

SUMMARY OF INVENTION

The present invention provides a method for reducing or inhibiting abiofilm comprising modulating expression of a cysB gene in a cellcapable of biofilm formation.

Further, the present invention provides a method for modulating theexpression of a cysB gene comprises contacting a cell capable of biofilmformation with a composition comprising a compound selected from thegroup consisting of ursolic acid or asiatic acid, or a pharmaceuticallyacceptable salt of such compound, a hydrate of such compound, a solvateof such compound, an N-oxide of such compound, or a combination thereof.

The present invention further provides a method for identifying a geneor genes involved in biofilm formation comprising a) mutating a gene,wherein the gene is a cysB gene or a gene related to cysB in at leastone cell capable of biofilm formation; b) contacting the cell with acompound selected from the group consisting of ursolic acid or asiaticacid or an analog of such compound; c) contacting at least one wild-typecell with the compound chosen in step b); and d) measuring the biofilmformation by the cell and the biofilm formation by the wild-type cell,wherein a modulation of the biofilm formation by the cell compared tothe biofilm formation by the wild-type cell indicates the involvement ofthe gene in biofilm formation.

The present invention provides a method for identifying an agent thatreduces or inhibits biofilm formation comprising contacting a cellcapable of biofilm formation with the agent; providing a reporter markerlinked to a gene, wherein the gene is a cysB gene or a gene related tocysB, wherein the reporter marker allows detection of the expression ofthe gene; and detecting modulation of the expression of the gene or ofits gene product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of biofilm formation in E. coli K12, P.aeruginosa PAO1, and V. harveyi BB120 with ursolic acid.

FIG. 2 shows the inhibition of air-liquid interface biofilm with ursolicacid.

FIG. 3 shows a comparison of the inhibition of biofilm formation bywild-type E. coli and mutant E. coli (cys B mutation) with ursolic acid.

FIGS. 4-8 show analogs of ursolic acid and asiatic acid.

FIG. 9 shows the chemical structures of ursolic acid and asiatic acid.

DESCRIPTION OF THE INVENTION Definitions

“Acceptable carrier” refers to a carrier that is compatible with theother ingredients of the formulation and is not deleterious to therecipient thereof.

“Reducing or inhibiting” in reference to a biofilm refers to theprevention of biofilm formation or growth, reduction in the rate ofbiofilm formation or growth, partial or complete inhibition of biofilmformation or growth.

“Modulates” or “modulating” refers to up-regulation or down-regulationof a gene's replication or expression.

Description

The present invention provides a method for reducing or inhibiting abiofilm comprising modulating the expression of a cysB gene in a cellcapable of biofilm formation.

Biofilm inhibitors can be used to treat diseases caused by bacteriaexisting in biofilms. For example, the inhibitors can contribute to thetreatment of cystic fibrosis. In cystic fibrosis, Pseudomonas aeroginosareside on the lungs of cystic fibrosis patients. The inhibitors canprevent, reduce, or eradicate the biofilm of Pseudomonas aeroginosa. Inaddition, biofilm inhibitors can prevent the attachment of Helicobactorpylori to gastric epithelial cells in patients with gastritis. Thisprevents the bacteria's invasion into these epithelial cells. Bypreventing H. pylori attachment to gastric epithelial cells, biofilminhibitors also prevent or reduce the risks associated with subsequentvirulence factors, such as arterial damage which may lead to a stroke.Moreover, biofilm inhibitors can also be used to treat urinary tractinfections. E. coli reside intracellularly in bladder cells. The E. coliresist conventional antibiotics and evade the host's immune systems. Thebiofilm inhibitors can prevent, control, reduce, or eradicate the E.coli. The biofilm inhibitors prevent or disrupt the attachment of E.coli to uroplakin or the proteins of the tight junctions of umbrellacells of the bladder, thereby potentially controlling the re-occurrenceof urinary tract infections.

Biofilm formation involves biological pathways conserved among differentspecies of bacteria. For example, different species of bacteria share acommon global regulator in the formation and maintenance of biofilms.Jackson et. al. showed catabolite repression induced by glucose caused30% to 95% reduction in biofilms among E. coli, Citrobacter freundii,Klebsiella pneumoniae, and Salmonella enterica Typhimurium. (Jackson, etal. J. Bacteriol. 2002, 184, 3406-3410). A bacterial autoinducer signal,AI-2, has been shown to be involved in the formation of biofilms. AI-2and genes responsive to this signal have been identified in a variety ofbacteria. Preferably, in an embodiment of the present invention, thebiofilm is reduced or inhibited by modulating expression of cysB inEscherichia coli, Proteus mirablis, Francisella tularensis, Vibrio sp.,Pseudomonas aeruginosa, V harveyi, Pseudomonas sp., Salmonella sp.,Haemophilus influenzae, Borrelia sp., Neisseria sp., Bacillus sp.,Burkholderia sp., Klebsiella sp., or Yersinia pestis. Still, preferably,the biofilm is reduced or inhibited by modulating expression of cysB ina Gram-negative bacteria.

CysB may be modulated in a number of ways. For example, N-acetyl-serineand sulfur limitation up-regulate cysB. Lochowska, A. et al., FunctionalDissection of the LysR-type CysB Transcriptional Regulator. J. Biol.Chem. 2001, 276, 2098-2107. In addition, like other LysR typeregulators, cysB can repress itself. Lilic, M. et al., Identification ofthe CysB-regulated gene, hslJ, related to the Escherichia colinovobiocin resistance phenotype. FEMS Micro. Letters. 2003, 224,239-246.

The disclosure herein describes another means to modulate cysB. Thepresent invention, therefore, also provides a method for modulating theexpression of a cysB gene comprising contacting the cell with acomposition comprising a compound selected from the group consisting ofursolic acid or asiatic acid, or a pharmaceutically acceptable salt ofsuch compound, a hydrate of such compound, a solvate of such compound,an N-oxide of such compound, or a combination thereof.

The disclosure herein describes the discovery that the cysB gene, atranscriptional regulator of the biosynthesis of cysteine, is involvedin biofilm formation. (Verschueren, K. H. G., Crystallization offull-length CysB of Klebsiella aerogenes, a LysR-type transcriptionalregulator, BIOLOGICAL CRYSTALLOGRAPHY D57:260-262, 2001). Asdemonstrated in the examples herein, the removal of cysB from E. coliresults in a significant reduction of biofilm formation in E. coli ascompared to wild-type E. coli. The cysB protein is a transcriptionalregulator of the LysR family of genes. The transcriptional regulators ofthis family have helix-turn-helix DNA binding motifs at theiramino-terminus. The cysB protein is required for the full expression ofthe cys genes, which is involved in the biosynthesis of cysteine.

The cysB gene is genetically conserved among different species ofbacteria, and more specifically Gram-negative bacteria. Verschueren, etal., Acta Cryst. (2001) D57, 260-262; Byrne et al., J. Bacteriol. 1988170 (7), p. 3150-3157. In fact, cysB is conserved among Pseudomonas sp.including, but not limited to, P. aeruginosa, P. putida, and P.syringae. (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi. Blastsearch of the cysB gene at the Microbial Genomics database at theNational Center for Biotechnology Information (NCBI) of the NationalInstitutes of Health (NIH)). The cysB gene is also genetically conservedamong the following species of bacteria: Vibrio sp. (e.g. V. harveyi andV. cholera), Proteus mirablis, Burkholderia sp. (e.g. B. fongorum, B.mallei, and B. cepacia), Klebsiella sp., Haemophilus influenza,Neisseria meningitides, Bordetella pertussis, Yersinia pestis,Salmonella typhimurium, and Acinetobacter sp.(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi. Blast search of thecysB gene at the Microbial Genomics database at NCBI of NIH). The cysBgene is also genetically conserved among the Gram-positive bacteria ofBacillus sp. including, but not limited to, B. subtilis, B. cereus, andB. anthracis. (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi. Blastsearch of the cysB gene at the Microbial Genomics database at NCBI ofNIH; van der Ploeg, J. R.; FEMS Microbiol. Lett. 2001, 201, p. 29-35).

In one embodiment of the present invention, the cell is selected from agroup consisting of Gram-negative bacteria. In another embodiment of theinvention, the cell is selected from a group consisting of Escherichiacoli, Proteus mirablis, Francisella tularensis, Vibrio sp., Pseudomonasaeruginosa, V. harveyi, Pseudomonas sp., Salmonella sp., Haemophilusinfluenzae, Borrelia sp., Neisseria sp., Bacillus sp., Burkholderia sp.,Klebsiella sp., and Yersinia pestis. Preferably, the cell is E. coli,Pseudomonas aeruginosa, or V. harveyi. As demonstrated in Example 2,ursolic acid reduces or inhibits the formation of biofilms in E. coli,P. aeruginosa, and V. harveyi. Using a similar method described inExample 2, asiatic acid was shown in Example 6 to reduce or inhibitbiofilm formation in E. coli.

Another embodiment of the present invention is a method for modulatingthe expression of cysD, cysI, cysJ, and/or cysK. Cys B controls thecysDIJK family of genes at the transcriptional level. Leyh, T., et al.J. Biol. Chem. 1992, 267(15), p. 10405-10410. Administration of ursolicacid down-regulates the expression of cysB and certain genes under itstranscriptional control, such as cysDIJK, while administration ofasiatic acid up-regulates the expression of cysB and certain genes underits transcriptional control. By modulating the expression of cysB,ursolic acid and asiatic acid reduce or inhibit biofilm formation.

Members of the family of LysR transcriptional regulators have beendemonstrated to regulate diverse metabolic processes. cysB exhibitsdirect control of the biosynthesis of cysteine. Verschueren et al., atp. 260. The cysB gene is involved, directly or indirectly, inglutathione intracellular transport, carbon source utilization, alaninedehydrogenases, and the arginine dependent system. YbiK is under thedirect control of cysB and participates in glutathione intracellulartransport. The data in example 1 demonstrates the down-regulation ofybiK by contacting a bacterial cell with ursolic acid. Thedown-regulation of ybiK in Example 1 of the specification furthersupports that ursolic acid down-regulates cysB. In an embodiment of theinvention, ursolic acid or asiatic acid modulates the expression ofybiK.

FIG. 9 shows the chemical structures of ursolic acid (C110) and asiaticacid (C255). Ursolic acid (UA) is a pentacyclic triterpene compoundisolated from many type of medicinal plants and is present in the humandiet. It has been reported to possess a wide range of pharmacologicalbenefits, including anti-cancer and anti-aging therapies. See e.g. Hsuet al., Life Sci. 75(19):2303-2316, Sep. 24, 2004 and Both et al., ArchDermatol. Res. 293(11):569-575, January 2002. Ursolic acid has also beenidentified as an antagonist for transforming growth factor (TGFβ1).Murakami et al., FEBS Lett. 566(1-3):55-59, May 21, 2004. However,before the disclosure herein, neither ursolic acid nor asiatic acid hasbeen reported to modulate the expression of the cysB gene. Neither haveursolic acid nor asiatic acid been reported to reduce or inhibit biofilmformations. Analogs of ursolic acid (C110) and asiatic acid (C255) areexpected to also modulate the expression of the cysB gene. FIGS. 4-8show examples of analogs of ursolic acid and asiatic acid.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.For parenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticgalactide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Another means to control biofilm formation is to understand theunderlying genetics involved. As it turns out, a complex web of genesregulates the formation and maintenance of biofilms by bacteria. Forinstance, Sauer et al. demonstrated that approximately 525 proteins aredifferentially regulated during the different stages of biofilmdevelopment in Pseudomonas aeruginosa. Sauer et al., J. Bacteriol.November 2004; 186(21):7312-26. Stanley et al. demonstrated thatapproximately 519 proteins are differentially regulated during the first24 hours of biofilm formation in Bacillus subtilis. Stanley, N. R. etal. J. Bacteriol. 2003, 185, 1951-1957. While numerous genes may beinvolved in a variety of biological pathways, only a few genes playcritical roles. Researchers spend considerable amount of effortdetermining which gene(s) are critical or essential in the biologicalpathways involved in various stages of biofilm formation andmaintenance. The disclosure herein describes the discovery of the genesinvolved in biofilm formation, such as cysB, cysD, cysI cysJ, cysKgene(s), and ybiK and the compounds that modulate these genes and reduceor inhibit biofilm formation.

Prior to the present invention, researchers look for genes involved inbiofilm formation by manipulating various factors, such as mediacondition, experimental temperature, random gene knock-out, and glucoselevel. These processes can be tedious, time-consuming, and costly. Seee.g. Sauer et al., J. Bacteriol. November 2004; 186(21):7312-26; Ren etal., Applied and Environmental Microbiology, April 2004,70(4):2038-2043; Verschueren et al., Biological Crystallography, 2001,D57:260-262; Pratt, L. A. and Roberto Kolter, Molecular Microbiology,October 1998, 30(2):285-293. The present invention provides a method foridentifying a gene or genes involved in biofilm formation comprising a)mutating a gene, wherein the gene is a cysB gene or a gene related tocysB in at least one cell capable of biofilm formation; b) contactingthe cell with a compound selected from the group consisting of ursolicacid or asiatic acid or an analog of such compound; c) contacting atleast one wild-type cell with the compound chosen in step b); and d)measuring the biofilm formation by the cell and the biofilm formation bythe wild-type cell, wherein a modulation of the biofilm formation by thecell compared to the biofilm formation by the wild-type cell indicatesthe involvement of the gene in biofilm formation.

As described herein, cysB is involved in biofilm formation. It controlsthe biosynthesis of cysteine. Verschureren et al., at p. 260. UsingcysB, standard methods can be used to identify other genes or geneproducts under its control that are involved in biofilm formation. Forexample, expression of cysB may be modulated while either modulating ormonitoring the expressions of the other genes suspected of beinginvolved in biofilm formation. This method identifies a gene's (or itsgene product) involvement in biofilm formation. A person of ordinaryskill in the art may perform additional tests to confirm the gene's (orits gene product) involvement in biofilm formation. Expressions ofeither cysB or genes under its control, such as cysDIJK family of genes,can be modulated, and using DNA microarrays (as demonstrated in theexamples) to determine direct or indirect effects as a result of themodulation. An inhibitor can also be used during these experiments topromote modulation of specific genes or gene products.

The present invention also provides a method for identifying novelagents that reduce or inhibit the formation of biofilms. As described inthe specification, the modulation of the expression of cysB inhibits theformation of biofilms. cysB is the global regulator of the biosynthesisof cysteine which directly controls the expression of the genes involvedin this process. The invention allows one skilled in the art ofscreening compounds in drug discovery to measure the modulation of agene, wherein the gene is a cysB gene or a gene related to cysB, duringthe screening of compounds as a novel detection method for the reductionor inhibition of biofilms. This method provides various advantages overcurrent screening strategies. Traditionally, the process of identifyingbiofilm inhibitors involves exposing at least one bacterial cell to acompound and then measuring the decrease in the formation of biofilms 24to 72 hours after exposure. The reduction in biofilm formation isquantified using crystal violet stain, which can be problematic. Asdescribed in the literature, after the bacteria are exposed to thecompounds, they are rinsed for a variable amount of time, stained for acertain amount of time with crystal violet stain, rinsed with solventsor combinations of solvents, and analyzed by determining opticaldensities of the crystal violet solutions compared to the controls.(Pratt, L. A. et al. Mol. Micro. 1998, 30(2), p. 285-298.). Therefore,measurement of the inhibition of biofilm formation can be laborious andcan yield unreliable results. Taking advantage of the discoverydescribed herein that modulation of a cysB gene is involved in biofilmformation, the present inventions provides a simple, fast, andinexpensive method of detecting the inhibition of biofilms. The methodinvolves the detection of the modulation of a gene, wherein the gene isa cysB gene or a gene related to cysB involved in biofilm formation. Areporter system is linked to the gene or its gene product. Specifically,the modulation of the cysB gene, a gene related to a cysB gene or itsgene product can be detected with a reporter, e.g., a green fluorescentprotein, antibiotic, radioactive isotope, or fluorescent dye.Accordingly, the present invention provides a superior method toidentify novel biofilm inhibitors than presently available in the art.

Biofilms may also adhere to surfaces, such as pipes and filters.Deleterious biofilms are problematic in industrial settings because theycause fouling and corrosion in systems such as heat exchangers, oilpipelines, and water systems. Elvers et al., Biofilms and Biofouling,2^(nd) ed., vol. 1, Academic Press, San Diego, Calif. Biofilm inhibitorscan be employed to prevent microorganisms from adhering to surfaceswhich may be porous, soft, hard, semi-soft, semi-hard, regenerating, ornon-regenerating. These surfaces include, but are not limited to,polyurethane, metal, alloy, or polymeric surfaces in medical devices,enamel of teeth, and cellular membranes in animals, preferably, mammals,more preferably, humans. The surfaces may be coated or impregnated withthe biofilm inhibitors prior to use. Alternatively, the surfaces may betreated with biofilm inhibitors to control, reduce, or eradicate themicroorganisms adhering to these surfaces.

The descriptions herein is not intended to limit the scope of thepresent invention, but only to demonstrate the far reaching utility ofthe invention to those skilled in the art. All references cited hereinare hereby incorporated by reference in their entirety.

EXAMPLES Example 1

Inhibition of biofilm formation by E. coli K12 [R1drd19], P. aeruginosaPAO1, and V. harveyi BB120 by the addition of 10 μg/mL ursolic acid. ForE. coli K12 [R1drd19], data were collected 16 hours after addition ofursolic acid to a 24 hour biofilm in LB medium; for P. aeruginosa PAO1,data were collected 18 hours after addition of ursolic acid withinoculation in LB medium plus 1% sodium citrate; and for V. harveyi BB120, data were collected 18 hours after addition of ursolic acid withinoculation in M9 medium. All biofilm mass readings at OD540 werenormalized based on the reading of wild type without ursolic acid whichwas normalized to 1. One standard deviation is shown. The results areshown in FIG. 1.

Example 2

Example 1 was repeated, except ursolic acid was added with inoculationin E. coli JM109 grown in LB 0.2% glucose. Ursolic acid inhibitedair-liquid interface biofilm. The results are shown in FIG. 2.

Example 3

To identify the genes controlled by ursolic acid, E. coli K12 was grownin LB medium overnight, diluted 1:100 in fresh LB supplemented with 0,10, or 30 μg/mL ursolic acid. The same amount of ethanol wassupplemented to eliminate solvent effects. The cultures were grown to anOD₆₀₀ of 0.9. The cells were centrifuged in a microcentrifuge for 15seconds at 20,000×g in mini bead beater tubes (Biospec, Bartlesville,Okla.) that were cooled to −80° C. before sampling. The cell pelletswere flash frozen in a dry ice-ethanol bath and stored at −80° C. untilRNA isolation.

To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, Calif.)and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to thefrozen bead beater tubes containing the cell pellets. The tubes wereclosed tightly and beat for 30 seconds at the maximum speed in a minibead beater (cat. no. 3110BX, Biospec). The total RNA was isolated byfollowing the protocol of the RNeasy Mini Kit (Qiagen) including anon-column DNase digestion with RNase-free DNase I (Qiagen). OD₂₆₀ wasused to quantify the RNA yield. OD₂₆₀/OD₂₈₀ and 23S/16S rRNA weremeasured using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto,Calif.) to check the purity and integrity of RNA (RNeasy Mini handbook,Qiagen).

The E. coli DNA microarrays were prepared as described previously byWei, Y. et al (Journal of Bacteriology, 2001, 183 (2) p. 545-556). Eachgene probe was synthesized by PCR and has the size of the full openreading frame (200-2000 nt). The double-strand PCR products weredenatured in 50% dimethyl sulfoxide and spotted onto aminosilane slides(Full Moon Biosystems, Sunnyvale, Calif.) as probes to hybridize withthe mRNA-derived cDNA samples. It has been shown that each array candetect 4228 of the 4290 E. coli ORFs. Each gene has two spots per slide.

Briefly, the total RNA from the E. coli K12 samples grown with andwithout ursolic acid was first converted into labeled cDNA. Then thecDNA samples (6 μg of each) were each labeled with both Cy3 and Cy5 dyesto remove artifacts related to different labeling efficiencies; hence,each experiment needed at least two slides. The Cy3-labeled samplewithout ursolic acid and the Cy5-labeled ursolic acid sample (with 10 or30 μg/mL ursolic acid) were hybridized on the first slide. Similarly,the Cy5-labeled sample without ursolic acid and the Cy3-labeled ursolicacid sample were hybridized on the second slide. Since each gene has twospots on a slide, the two hybridizations generated eight data points foreach gene (four points for the sample without ursolic acid and fourpoints for the ursolic acid sample). The microarray experiments withdye-swapping were repeated for both concentrations of ursolic acid.

The cDNA samples of E. coli DH5α treated with FCM or 0.5× LB (6 μg ofeach) were each labeled with both Cy3 and Cy5 dyes to remove artifactsrelated to different labeling efficiencies; hence, each experimentrequired at least two slides. The Cy3-labeled FCM sample and Cy5-labeled0.5× LB sample were hybridized on the first slide. Similarly, theCy5-labeled FCM sample and Cy3-labeled 0.5× LB sample were hybridized onthe second slide. Since each gene has two spots on a slide, the twohybridizations generated eight data points for each gene (four pointsfor the FCM sample and four points for the 0.5× LB sample). DNAmicroarrays for the E. coli DH5α treated with ACM or 0.5× LB wereperformed in an analogous manner.

The DNA microarrays were incubated in prehybridization solution (3.5×SSC[1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate] [Invitrogen], 0.1%sodium dodecyl sulfate [SDS] [Invitrogen], and 0.1% bovine serum albumin[Invitrogen]) at 45° C. for 20 min. The arrays were rinsed withdouble-distilled water and were spun dry by centrifugation. Labeled cDNA(6 μg) was concentrated to 10 μl of total volume and was mixed with 10μl of 4× cDNA hybridization solution (Full Moon Biosystems) and 20 μl offormamide (EM Science, Gibbstown, N.J.). The hybridization mix washeated to 95° C. for 2 min and was added to the DNA microarrays; eacharray was covered with a coverslip (Corning, Big Flats, N.Y.) and wasincubated overnight at 37° C. for hybridization. When the hybridizationwas finished, the coverslips were removed in 1×SSC-0.1% SDS at roomtemperature, and the arrays were washed once for 5 min in 1×SSC-0.1% SDSat 40° C., twice for 10 min in 0.1×SSC-0.1% SDS at 40° C., and twice for1 min in 0.1×SSC at 40° C. The arrays were quickly rinsed by dipping inroom-temperature double-distilled water and were then spun dry bycentrifugation. The hybridized slides were scanned with the GenerationIII Array Scanner (Molecular Dynamics Corp.). Readings at 570 and 670 nmwas used to quantify the probes labeled with Cy3 and Cy5 separately. Thesignal was quantified with Array Vision 4.0 or 6.0 software (ImagingResearch, St. Catherines, Ontario, Canada). Genes were identified asdifferentially expressed if the expression ratio was greater than 1.4and the p-value (t-test) is less than 0.05. P-values were calculated onlog-transformed, normalized intensities. Including the p-value criterionensures the reliability of the induced/repressed gene list.Normalization was relative to the median total fluorescent intensity perslide per channel.

TABLE 1 E. coli K12 genes repressed by 10 and 30 μg/mL ursolic acid. Theunderlined ratios indicate the corresponding genes were significantlyrepressed by ursolic acid. The highlighted genes were repressed both by10 and 30 μg/mL ursolic acid. ER is expression ratio and Pv is p-value.10 μg/mL 30 μg/mL ursolic acid ursolic acid Gene b# ER Pv ER PvDescription arsC b3503 −1.5 0.045   1.4 0.014 enzyme, drug/analogsensitivity b2789 b2789 −1.9 0.056 −2.5 0.038 putative D-glucaratepermease (MFS family) cspF b1558 −1.6 0.003 −1.1 0.357 cold shock-likeprotein cspG b0990 −2.5 0.009 −1.7 0.017 homolog of Salmonella coldshock protein cysB b1275 −1.7 0.038 −1.4 0.018 positive transcriptionalregulator for cysteine regulon cysD b2752 −2.5 9E−04 −2.5 0.024 centralintermediary metabolism: sulfur metabolism cysI b2763 −1.5 0.069 −1.72E−04 central intermediary metabolism: sulfur metabolism cysJ b2764 −3.60.015 −3.3 0.009 central intermediary metabolism: sulfur metabolism cysKb2414 −3.6 0.003 −3.3 0.008 amino acid biosynthesis: cysteine frvR b3897−5.4 0.006 −2   0.175 putative frv operon regulatory protein gntU_1b3436 −1.5 0.026 −1.4 0.043 transport of small molecules: carbohydrates,organic acids, alcohols narH b1225 −1.6 0.002 −1.4 0.028 energymetabolism, carbon: anaerobic respiration pheM b1715 −1.6 0.011 1  0.762aminoacyl tRNA synthetases, tRNA modification pheP b0576 −1.5 0.021 −1.10.499 transport of small molecules: amino acids, amines rimL b1427 −1.50.022 1  0.719 enzyme, ribosomes - maturation and modification rmf b0953−1.5 0.003 1  0.662 factor; ribosomes - maturation and modification rpmIb1717 −1.6 0.007 1  0.708 structural component, ribosomal proteins -synthesis, modification slp b3506 −1.5 0.006 −1.6 0.002 outer membraneconstituents ugpB b3453 −1.4 0.045 −1.5 0.021 transport of smallmolecules: carbohydrates, organic acids, alcohols ybiK b0828 −2.4 7E−04−2.2 0.005 putative asparaginase yhaD b3124 −1.6 0.025 −2.6 0.009glycerate kinase I yhaF b3126 −1.5 0.009 −2.4 0.002alpha-dehydro-beta-deoxy-D-glucarate aldolase yhaG b3128 −2   0.004 −2.20.008 (D)-galactarate dehydrogenase b0309 b0309 −1.7 0.042 −1.3 0.155orf, hypothetical protein b0484 b0484 −1.5 0.044 −1.1 0.425 putativeenzyme, not classified b0485 b0485 −1.8 0.009 −1.3 0.019 putativeenzyme, not classified b0829 b0829 −1.5 0.032 −1.5 0.09 putativetransport; not classified b1729 b1729 −5.6 0.003 −2   0.133 putativeenzyme, not classified b2379 b2379 −1.5 0.011 1  0.325 putative enzyme,not classified hdeA b3510 −1.7 0.008 −1.4 0.008 orf, hypotheticalprotein hdeB b3509 −1.8 6E−04 −1.4 0.01 orf, hypothetical protein yeeDb2012 −2.3 0.025 −1.4 0.228 orf, hypothetical protein yeeE b2013 −13  0.006 −2   0.182 putative transport, not classified yjeB b4178 −1.40.005 1  0.833 orf, hypothetical protein ybhG b0795 −1.4 0.013 −1.40.002 putative membrane, not classified yhaU b3127 −1.9 0.074 −4.2 0.003putative transport protein

Example 4

Effect of adding 30 μg/mL ursolic acid on biofilm formation in LB mediumin the presence the cysB mutation (E. coli K12 [R1drd19] vs. E. coli K12cysB[R1drd19], data collected 16 hours after addition of ursolic acid.All biofilm mass readings at OD540 were normalized based on the readingof wild type without ursolic acid which was normalized to 1. Onestandard deviation shown. The results are shown in FIG. 3.

Example 5

Example 1 was repeated, except asiatic acid was added with inoculationin E. coli JM109 in MC9 glucose media. Asiatic acid demonstratedapproximately 75%, 80%, and 85% biofilm inhibition when tested at 5μg/ml, 10 μg/ml, and 15 μg/ml, respectively.

Example 6

Example 3 was repeated, except asiatic acid (C255) was added instead ofursolic acid. The results are shown in Table 2.

TABLE 2 E. coli JM109 genes induced by 10 μg/ml and 30 μg/ml asiaticacid in M9C glucose media. The underlying ratios indicate thecorresponding genes were significantly induced by asiatic acid. ER isexpression ratio and Pv is p-value. 10 ug/ml 30 ug/ml Asiatic AcidAsiatic Acid Gene b# Pv ER Pv ER Descriptions b0829 b0829 0.000001 2.830.000001 2.46 putative ATP-binding component of a transport system b1729b1729 0.000001 2.64 0.000001 2.30 part of a kinase b1963 b1963 0.0008382.14 0.000007 2.30 orf, hypothetical protein b2332 b2332 0.000002 2.140.000002 2.30 orf, hypothetical protein b2420 b2420 0.000006 4.290.004108 2.14 orf, hypothetical protein b2531 b2531 0.000002 2.460.000002 2.14 orf, hypothetical protein b2670 b2670 0.000001 3.250.002057 2.64 orf, hypothetical protein b2834 b2834 0.000063 2.300.000121 2.00 orf, hypothetical protein bolA b0435 0.000001 2.300.000001 2.64 possible regulator of murein genes cbl b1987 0.00000113.93 0.000001 13.00 transcriptional regulator cys regulon; accessoryregulatory circuit affecting cysM cysA b2422 0.000002 4.00 0.000002 3.48ATP-binding component of sulfate permease A protein; chromate resistancecysB b1275 0.000001 6.06 0.000001 4.29 positive transcriptionalregulator for cysteine regulon cysC b2750 0.000001 10.56 0.000001 6.96adenosine 5-phosphosulfate kinase cysD b2752 0.000001 6.96 0.000001 6.50ATP: sulfurylase (ATP: sulfate adenylyltransferase), subunit 2 cysHb2762 0.000001 4.59 0.000001 3.03 3-phosphoadenosine 5-phosphosulfatereductase cysI b2763 0.000002 4.29 0.000002 3.25 sulfite reductase,alpha subunit cysJ b2764 0.000002 3.73 0.000002 4.00 sulfite reductase(NADPH), flavoprotein beta subunit cysK b2414 0.000001 4.59 0.0000013.48 cysteine synthase A, O-acetylserine sulfhydrolase A cysM b24210.000001 3.73 0.000004 3.25 cysteine synthase B, O-acetylserinesulfhydrolase B cysN b2751 0.000001 11.31 0.000001 7.46 ATP-sulfurylase(ATP: sulfate adenylyltransferase), subunit 1, probably a GTPase cysPb2425 0.000001 4.29 0.000001 4.92 thiosulfate binding protein cysU b24240.000002 4.92 0.000002 4.92 sulfate, thiosulfate transport systempermease T protein cysW b2423 0.000002 4.59 0.000002 4.00 sulfatetransport system permease W protein dgt b0160 0.000002 2.14 0.0000572.30 deoxyguanosine triphosphate triphosphohydrolase fliY b1920 0.0000024.00 0.000002 3.25 putative periplasmic binding transport protein ftnb1905 0.000001 3.25 0.000001 3.03 cytoplasmic ferritin (an iron storageprotein) glgS b3049 0.000001 2.14 0.000001 2.30 glycogen biosynthesis,rpoS dependent ilvG_1 b3767 0.000005 2.83 0.000059 2.00 acetolactatesynthase II, large subunit, cryptic, interrupted ilvL b3766 0.0000032.30 0.000005 3.48 ilvGEDA operon leader peptide msrA b4219 0.0000012.30 0.000001 2.64 peptide methionine sulfoxide reductase nlpA b36610.000001 18.38 0.000001 9.85 lipoprotein-28 pssR b3763 0.001336 2.460.001832 2.14 regulator of pssA pstS b3728 0.000002 2.64 0.000002 2.00high-affinity phosphate-specific transport system; periplasmicphosphate-binding protein sbp b3917 0.000001 18.38 0.000001 12.13periplasmic sulfate-binding protein tauA b3065 0.000001 2.46 0.0000012.00 taurine transport system periplasmic protein yaeG b0162 0.0073984.59 0.042948 4.00 orf, hypothetical protein yaiB b0382 0.000001 2.300.000014 2.64 orf, hypothetical protein ybgR b0752 0.000002 2.460.000002 2.46 putative transport system permease protein ybiK b08280.000001 6.50 0.000001 5.66 putative asparaginase yciW b1287 0.0000023.03 0.000002 3.73 putative oxidoreductase yedO b1919 0.000002 3.730.000002 2.83 putative 1-aminocyclopropane-1-carboxylate deaminase yeeDb2012 0.000002 3.73 0.000002 2.30 orf, hypothetical protein yeeE b20130.000002 3.25 0.000002 2.14 putative transport system permease proteinygbE b2749 0.000002 6.96 0.000004 5.28 putative cytochrome oxidasesubunit yicG b3646 0.000001 2.83 0.000001 2.30 orf, hypothetical proteinyicL b3660 0.000001 6.50 0.000057 2.83 putative permease transporteryjaE b3995 0.000002 2.00 0.000002 2.14 putative transcriptionalregulator yjiD b4326 0.000001 7.46 0.000001 9.85 orf, hypotheticalprotein yrbL b3207 0.000001 2.64 0.000001 2.46 orf, hypothetical protein

1. A method for reducing or inhibiting a biofilm comprising modulatingexpression of a gene present in a Gram-negative bacteria cell capable ofbiofilm formation, the gene being a cysD, cysI, cysJ, cysK or ybiK gene,thereby to reduce or inhibit the biofilm formation.
 2. The method ofclaim 1, wherein modulation of the gene comprises contacting the cellwith a composition comprising a compound selected from the groupconsisting of ursolic acid and asiatic acid, or a pharmaceuticallyacceptable salt of such compound, a hydrate of such compound, a solvateof such compound, an N-oxide of such compound, or a combination thereof.3. The method of claim 2, wherein the compound is ursolic acid.
 4. Themethod of claim 2, wherein the compound is asiatic acid.
 5. The methodof claim 1, wherein the cell is selected from a group consisting ofEscherichia coli, Proteus mirablis, Francisella tularensis, Vibrio sp.,Pseudomonas aeruginosa, V. harveyi, Pseudomonas sp., Salmonella sp.,Haemophilus influenzae, Borrelia sp., Neisseria sp., Bacillus sp.,Burkholderia sp., Klebsiella sp., and Yersinia pestis.
 6. The method ofclaim 5, wherein the cell is Escherichia coli.
 7. The method of claim 5,wherein the cell is Pseudomonas aeruginosa.
 8. The method of claim 5,wherein the cell is V. harveyi.
 9. (canceled)
 10. The method of claim 2,wherein the gene is cysD.
 11. The method of claim 2, wherein the gene iscysI.
 12. The method of claim 2, wherein the gene is cysJ.
 13. Themethod of claim 2, wherein the gene is cysK.
 14. The method of claim 2,wherein the gene is ybiK.
 15. A method for modulating the expression ofa gene selected from the group consisting of cysD, cysI, cysK or ybiK,comprising contacting a Gram-negative bacteria cell capable of biofilmformation with a composition comprising a compound selected from thegroup consisting of ursolic acid and asiatic acid, or a pharmaceuticallyacceptable salt of such compound, a hydrate of such compound, a solvateof such compound, a N-oxide of such compound, or a combination thereof.16. The method of claim 15, wherein the compound is ursolic acid. 17.The method of claim 15, wherein the compound is asiatic acid.
 18. Themethod of claim 15, wherein the gene is cysD.
 19. The method of claim15, wherein the gene is cysI.
 20. The method of claim 15, wherein geneis cysJ.
 21. The method of claim 15, wherein the gene is cysK.
 22. Themethod of claim 15, wherein the gene is ybiK.
 23. The method of claim15, wherein the cell is selected from a group consisting of Escherichiacoli, Proteus mirablis, Francisella tularensis, Vibrio sp., Pseudomonasaeruginosa, V. harveyi, Pseudomonas sp., Salmonella sp., Haemophilusinfluenzae, Borrelia sp., Neisseria sp., Bacillus sp., Burkholderia sp.,Klebsiella sp., and Yersinia pestis.
 24. The method of claim 23, whereinthe cell is Escherichia coli.
 25. The method of claim 23, wherein thecell is Pseudomonas aeruginosa.
 26. The method of claim 23, wherein thecell is V. harveyi.
 27. The method of claim 15, wherein the cell isselected from a group consisting of Gram-negative bacteria. 28.-38.(canceled)